Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway.
Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway.
复制标题
DOI：
10.1371/journal.pone.0082511
复制
发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Freedman RB
中科院分区：
文献类型：
--
作者：
Irvine AG;Wallis AK;Sanghera N;Rowe ML;Ruddock LW;Howard MJ;Williamson RA;Blindauer CA;Freedman RB
与分子伴侣蛋白将蛋白质折叠与ATP水解偶联不同，蛋白质二硫异构酶（PDI）催化蛋白质折叠偶联到二硫键形成（氧化折叠）。然而，我们不知道PDI如何区分折叠的、部分折叠的和未折叠的蛋白质底物。作为氧化折叠途径的模型中间体，我们制备了碱性胰蛋白酶抑制剂（BPTI）的双二硫突变体，并通过核磁共振证明其部分折叠且高度动态。核磁共振研究表明，它在与肽配体结合的同一位点与PDI结合，具有快速的结合和解离动力学；表面等离子体共振显示其与PDI的相互作用Kd约为10−5 m。为了比较，我们表征了PDI与天然BPTI和完全展开的BPTI的相互作用。有趣的是，PDI确实与天然BPTI结合，但与部分折叠和未折叠的BPTI相比，这种结合在数量上弱。因此PDI通过永久或瞬时展开的区域识别和结合底物。这是第一次研究PDI与部分折叠蛋白质的相互作用，也是第一次分析这种折叠催化剂沿着氧化折叠途径与底物变化的相互作用。我们已经确定了使PDI成为氧化蛋白折叠的有效催化剂的关键特征-差异亲和力，快速配体交换和构象灵活性。
In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10−5 M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility.
登录
查看更多内容
影响因子：
5.6
作者：
CREIGHTON, TE;BAGLEY, CJ;SHEIKH, A
通讯作者：
SHEIKH, A
影响因子：
4.8
作者：
Masui, Shoji;Vavassori, Stefano;Inaba, Kenji
通讯作者：
Inaba, Kenji
影响因子：
5.6
作者：
DARBY, NJ;MORIN, PE;CREIGHTON, TE
通讯作者：
CREIGHTON, TE
影响因子：
4.8
作者：
Pirneskoski, A;Klappa, P;Ruddock, LW
通讯作者：
Ruddock, LW
DOI：
10.1073/pnas.47.9.1309
发表时间：
1961-01-01
影响因子：
11.1
作者：
ANFINSEN, CB;HABER, E;WHITE, FH
通讯作者：
WHITE, FH